Biology subjects

Bhatia, S.

Publications and source records attributed to Bhatia, S..

53 records · Page 3Linked to original sources

bioRxiv2020

Effects of NT5C2 germline variants on 6-mecaptopurine metabolism in children with acute lymphoblastic leukemia

6-mercaptopurine (6-MP) is widely used in the treatment of acute lymphoblastic leukemia (ALL), and its cytotoxicity is primarily mediated by thioguanine nucleotide metabolites (TGN). Recent genome-wide association study has identified germline polymorphisms (e.g., rs72846714) in the NT5C2 gene associated with 6-MP metabolism in patients with ALL. However, the full spectrum of genetic variation in NT5C2 is unclear and its impact on 6-MP drug activation has not been comprehensively examined. To this end, we performed targeted sequencing of NT5C2 in 588 children with ALL and identified 121 single nucleotide polymorphisms (SNPs) nominally associated with erythrocyte TGN during 6-MP treatment (P < 0.05). Of these, 61 variants were validated in a replication cohort of 372 children with ALL. After considering linkage disequilibrium and multivariate analysis, we confirmed two clusters of variants, represented by rs72846714 and rs58700372, that independently affected 6-MP metabolism. Functional studies showed that rs58700372 directly altered the activity of an intronic enhancer, with the variant allele linked to higher transcription activity and reduced 6-MP metabolism (lower TGN). By contrast, rs72846714 was not located in a regulatory element and instead its association signal was explained by linkage disequilibrium with a proximal functional variant rs12256506 that activated NT5C2 transcription in-cis. Our results indicated that NT5C2 germline variation significantly contributes to inter-patient variability in thiopurine drug disposition.

genetics↗

bioRxiv2020

Q-STARZ: Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish

Noncoding regions of the genome harbouring cis-regulatory elements (CREs) or enhancers drive spatial and temporal gene expression. Mutations or single nucleotide polymorphisms (SNPs) in enhancers have been widely implicated in human diseases and disease-predispositions. However, our ability to assay the regulatory potential of genetic variants in enhancers is currently very limited, in part because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type (Wt) and disease-associated, mutant (Mut) human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. Using this single transgenic cassette, the functional activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing the simultaneous visualisation of the activity of both alleles. This can reveal where and when in embryonic development the wild-type allele is active and how this activity is altered by the disease-associated mutation.

genetics↗

bioRxiv2020

Functional Predictors of Causative Cis-Regulatory Mutations in Mendelian Disease

Undiagnosed neurodevelopmental disease is significantly associated with rare variants in cis-regulatory elements (CRE) but demonstrating causality is challenging as target gene consequences may differ from a causative variant affecting the coding region. Here, we address this challenge by applying a procedure to discriminate likely diagnostic regulatory variants from those of neutral/low-penetrant effect. We identified six rare CRE variants using targeted and whole genome sequencing in 48 unrelated males with apparent X-linked intellectual disability (XLID) but without detectable coding region variants. These variants segregated appropriately in families and altered conserved bases in predicted CRE targeting known XLID genes. Three were unique and three were rare but too common to be plausibly causative for XLID. We compared the cis-regulatory activity of wild-type and mutant alleles in zebrafish embryos using dual-color fluorescent reporters. Two variants showed striking changes: one plausibly causative (FMR1CRE) and the other likely neutral/low-penetrant (TENM1CRE). These variants were "knocked-in" to mice and both altered embryonic neural expression of their target gene. Only Fmr1CRE mice showed disease-relevant behavioral defects. FMR1CRE is plausibly disease-associated resulting in complex misregulation of Fmr1/FMRP rather than loss-of-function. This is consistent both with absence of Fragile X syndrome in the probands and the observed electrophysiological anomalies in the FMR1CRE mouse brain. Although disruption of in vivo patterns of endogenous gene expression in disease-relevant tissues by CRE variants cannot be used as strong evidence for Mendelian disease association, in conjunction with extreme rarity in human populations and with relevant knock-in mouse phenotypes, such variants can become likely pathogenic.

genetics↗

bioRxiv2020

CRISPR-Cas-amplified urine biomarkers for multiplexed and portable cancer diagnostics

Synthetic biomarkers, exogenous probes that generate molecular reporters, represent an emerging paradigm in precision diagnostics with applications across infectious and noncommunicable diseases. In order to achieve their promise, these methods reply on multiplexing strategies to provide tools that are both sensitive and specific. However, the field of synthetic biomarkers has not benefited from molecular strategies such as DNA-barcoding due to the susceptibility of nucleic acids in vivo. Herein, we exploit chemically-stabilized DNAs to tag synthetic biomarkers and produce diagnostic signals via CRISPR nucleases. Our strategy capitalizes on disease-associated, protease-activated release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed biofluids. In murine cancer models, we show that the DNA-encoded urine biomarkers can noninvasively detect and monitor disease progression, and demonstrate that nuclease amplification can be harnessed to convert the readout to a point-of-care tool. This technique combines specificity with ease of use to offer a new platform to study human disease and guide therapeutic decisions.

bioengineering↗

bioRxiv2020

A New Form of Decellularized Extracellular Matrix Hydrogel for Treating Ischemic Tissue via Intravascular Infusion

Biomaterials, such as extracellular matrix (ECM) hydrogels, have been widely used in preclinical studies as injectable tissue engineering therapies; however, injectable therapies are limited as they can cause localized trauma or organ perforation. We have developed a new ECM therapy, the low molecular weight fraction derived from decellularized, digested ECM, for intravascular infusion. This new form of ECM can be infused after injury, specifically localize to injured tissues by coating the leaky microvasculature, and promote cell survival and tissue repair. In this study, we show the feasibility and targeting of intravascular ECM infusions using models of acute myocardial infarction (MI), traumatic brain injury, and pulmonary arterial hypertension. Furthermore, safety and efficacy were demonstrated in small and large animal models of acute MI following intracoronary infusion, which included using a clinically-relevant catheter in the large animal model. Functional improvements, specifically reduced left ventricular volumes and improved wall motion scores were observed after ECM infusions post-MI. Genes related to tissue repair and inflammation were differential expressed in response to ECM infusions. This study shows proof-of-concept for a new paradigm of delivering pro-healing ECM biomaterials via intravascular infusion to heal tissue from the inside out.

bioengineering↗

bioRxiv2020

MaTAR25 LncRNA Regulates the Tensin1 Gene to Impact Breast Cancer Progression

Misregulation of long non-coding RNA genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin 1 (Tns1) to regulate its expression in trans. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. The human ortholog of MaTAR25, LINC01271, is upregulated with human breast cancer stage and metastasis. SIGNIFICANCELncRNAs have great potential to reveal new regulatory mechanisms of function as well as having exciting therapeutic capacity given their ease of being targeted by nucleic acid drugs. Our study of MaTAR25, and its human ortholog LINC01271, reveal an unexpected function of this lncRNA in breast cancer progression by regulating Tns1 gene expression, whose protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. We identified LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and cancer metastasis. Our findings demonstrate that LINC01271 represents an exciting therapeutic target to alter breast cancer progression.

cancer biology↗

bioRxiv2020

Generalizability of "GWAS hits" in clinical populations: Lessons from childhood cancer survivors

With mounting interest in translating GWAS hits from large meta-analyses (meta-GWAS) in diverse clinical settings, evaluating their generalizability in target populations is crucial. Here we consider long-term survivors of childhood cancers from the St. Jude Lifetime Cohort Study and show the limited generalizability of 1,376 robust SNP associations reported in the general population across 12 complex anthropometric and cardiometabolic phenotypes (N=2,231; observed-to-expected replication ratio=0.68, P=2.4x10-9). An examination of five comparable phenotypes in a second independent cohort of survivors from the Childhood Cancer Survivor Study corroborated the overall limited generalizability of meta-GWAS hits to survivors (N=4,212, observed-to-expected replication ratio=0.53, P=1.1x10-16). Meta-GWAS hits were less likely to be replicated in survivors exposed to cancer therapies associated with phenotype risk. Examination of complementary DNA methylation data in a subset of survivors revealed that treatment-related methylation patterns at genomic sites linked to meta-GWAS hits may disrupt established genetic signals in survivors.

genetics↗

bioRxiv2020

Identification of miRNAs and their corresponding mRNA targets from chickpea root infected with M. ciceri and functional characterization of candidate miRNAs by overexpression in chickpea roots

O_LILegumes developed symbiotic associations with rhizobia to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. C_LIO_LIOne microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced to unravel the miRNA mediated regulation of symbiosis in chickpea. C_LIO_LISequencing of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. C_LIO_LIPhylogenetic analysis of the precursor sequences of the 91 miRNAs clearly indicated a clustering of two distinct miRNAs in the same clade representing a close ancestral precursor. C_LIO_LIFurther, biogenesis of miRNAs was predicted using the miRNAs identified from different legume genomes. C_LIO_LIThe miRNA reads from the nodule library were also mapped onto bacterial genomes from which bacterial small RNA were predicted. C_LIO_LIThe antagonistic expression of some of the miRNA-target pairs was investigated and the negative co-related expression profiling proved the validity of the libraries and the miRNA-target pairs. Four miRNAs were selected based on the antagonistic expression profiling and were ectopically expressed in chickpea roots by hairy root transformation. C_LIO_LIThe overexpression lines showed significant change in nodule numbers. The target of miR171f (NRK), miR394 (HP) and miR1509 (AK) are novel ones being reported for the first time. This analysis opens a wide arena for investigation of the novel miRNAs and target pairs, polycistronic miRNAs and the bacterial derived smRNAs predicted in this study. C_LI

plant biology↗

bioRxiv2019

Three complete genome sequences of Penguin megrivirus from ornithogenic soil, Adelie penguin and Weddell seal of Antarctica

We report the complete genome sequences of Penguin megriviruses from three different sources from Antarctica namely feces of Adelie penguin, feces of Weddell seal and ornithogenic soil. Phylogenetic analysis indicates the prevalence of very similar viruses in different sources of Antarctic environment. These genome sequences aid to understand the evolution of megriviruses in Antarctic ecology and reveal their place in global megrivirus phylogeny.

microbiology↗

bioRxiv2019

Comprehensive analysis of structural variants in breast cancer genomes using single molecule sequencing

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of disease progression. We performed whole genome sequencing of the SKBR3 breast cancer cell-line and patient-derived tumor and normal organoids from two breast cancer patients using 10X/Illumina, PacBio, and Oxford Nanopore sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings demonstrate that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long-reads even at relatively low coverage (25x-30x). Furthermore, we inferred karyotypes from these data using our enhanced RCK algorithm to present a more accurate representation of the mutated cancer genomes, and find hundreds of variants affecting known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

bioinformatics↗

bioRxiv2019

Comprehensive molecular characterization of pediatric treatment-induced high-grade glioma: A distinct entity despite disparate etiologies with defining molecular characteristics and potential therapeutic targets

Treatment-induced high-grade gliomas (TIHGGs) are an incurable late complication of cranial radiation therapy or combined radiation/chemotherapy used to treat pediatric cancer. We assembled a cohort of 33 TIHGGs from multiple institutions. The primary antecedent malignancies were medulloblastoma, acute lymphoblastic leukemia, astrocytoma, and ependymoma. We performed methylation profiling, RNA-seq, and genomic sequencing (whole-genome or whole-exome) on TIHGG samples. Methylation profiling revealed that TIHGGs cluster primarily with the pediatric receptor tyrosine kinase I subtype (26/31 samples). Common TIHGG copy-number alterations include Chromosome (Ch.) 1p loss/1q gain, Ch. 4 loss, Ch. 6q loss, and Ch. 13 and Ch. 14 loss; focal alterations include PDGFRA and CDK4 gain and loss of CDKN2A and BCOR. Relative to de novo pediatric high-grade glioma (pHGG), BCOR loss (p=0.004) and CDKN2A loss (p=0.005) were significantly increased. Transcriptomic analysis identified two distinct TIHGG subgroups, one with a lesser mutation burden (0.12 mut/Mb), Ch. 1p loss/1q gain (5/6 samples), and stem cell characteristics, and one with a greater mutation burden (1.08 mut/Mb, p<0.0002), depletion of DNA repair pathways, and inflammatory characteristics. We observed increased chromothripsis in TIHGG versus pHGG (67% vs. 31%, p=0.036), which was associated with extrachromosomal circular DNA-mediated amplification of PDGFRA and CDK4. In vitro drug screening in one primary, patient-derived TIHGG cell line from each expression subgroup identified microtubule inhibitors/stabilizers, DNA-damaging agents, MEK inhibition, and, in the inflammatory subgroup, proteasome inhibitors as potentially effective therapies. This study provides a comprehensive molecular profile of TIHGG, including mechanistic insights to TIHGG oncogenesis, and identifies potentially effective therapeutic modalities for further investigation.

cancer biology↗

bioRxiv2019

Massive parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino-acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

genomics↗

bioRxiv2019

SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans

RNA interference (RNAi) is sequence-specific gene silencing triggered by double-stranded (ds)RNA. When dsRNA is expressed or introduced into one cell and is transported to and initiates RNAi in other cells, it is called systemic RNAi. Systemic RNAi is very efficient in C. elegans and genetic screens for systemic RNAi defective (Sid) mutants have identified RNA transporters (SID-1, SID-2 and SID-5) and a signaling protein (SID-3). Here we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dosesensitive, systemic RNAi defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in dsRNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting they may function in a common signaling pathway, however, a sid-3; sid-4 double mutants showed additive resistance to RNAi, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNAi sensitivity tests identified 23 additional signaling components with weak RNAi defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNAi efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNAi silencing efficiency.

genetics↗

bioRxiv2019

Global analysis of Two Component System (TCS) member of chickpea and other legume genomes implicates its role in enhanced nodulation

Nodule organogenesis is governed primarily by phytohormone cytokinin. We observed the significant nodulation in chickpea at particular cytokinin concentration (2.5x10-7) which indicated the importance of cytokinin in nodule development. Cytokinin signaling is mediated through the Two Component System (TCS) which comprises of sensor histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs). Therefore, we analyzed the interconnection of cytokinin with TCS molecules during root nodule development through global analysis of TCS candidates in legumes with special consideration to cytokinin receptor and Type-B RR member. We have conducted an in depth global analysis of TCS family members in chickpea and other legumes, Medicago and pigeon pea. Higher number of TCS genes were found in Medicago (96), followed by pigeonpea (75) and chickpea (67). A good correlation between TCS members with their corresponding total number of genes were observed in all three-legume species. Collinearity analysis of TCS revealed phylogenetically closer proximity of Cicer to Medicago followed by Glycine than Cajanus. Comprehensive analysis of 3-dimensional structure, genomic organisation and domain arrangement showed a conservation of TCS members within species. In depth investigation showed that HKs were mainly conserved among TCS members in legumes and non-legumes while divergence occurred at level of RRs. Further, Type-B RRs were functionally most diversified in RRs based on phylogeny, syntenic and transcript analysis. Few numbers of segmentally duplicated pair of TCS showed difference in their transcriptional regulation suggesting the functional evolution. For functional characterization the cre1 mutants of (Medicago) were complemented with chickpea cytokinin responsive HKs and nodulation deficient phenotype of mutants were restored. A synchronous cytokinin-induced expression of chickpea cytokinin receptor HKs and CaNIN provides strong relation of cytokinin signaling during nodulation. Furthermore, interesting potential candidate CaRR13 was selected to deduce the underlying molecular mechanism of nodulation, chickpea in specific and legumes in general.

genomics↗

bioRxiv2019

Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

-Synuclein (-Syn) aggregation and amyloid formation is directly linked with Parkinsons disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of -Syn precedes its aggregation. In particular, in vitro generated -Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate -Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote -Syn LLPS and its subsequent maturation. We further demonstrate -Syn liquid droplet formation in cells, under oxidative stress. These cellular -Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured -Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

neuroscience↗

bioRxiv2018

Identification of a novel tetrameric structure for human apolipoprotein-D

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded {beta}-barrel with a single adjacent -helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimers disease, where apolipoprotein-D overexpression reduces the amyloid-{beta} burden in Alzheimers disease mouse models.\n\nOligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.\n\nHere, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a {bsim}95 to {bsim}100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

biochemistry↗

bioRxiv2018

Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. We identify 1,079 PTEN and 805 TPMT single amino acid variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and our abundance data suggest that a PTEN variant accounting for ~10% of PTEN missense variants in melanomas functions via a dominant negative mechanism. Finally, we demonstrate that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable assay for characterizing missense variants.

genetics↗